Ehrlichia

The partial nucleotide sequences of microorganisms obtained for 18S rRNA, 16S rRNA and citrate synthase (gltA) genes were used to assess the genetic relationships with those of species of Babesia/Theileria, Ehrlichia and Rickettsia, respectively. Reference sequences were downloaded for each pathogen from GenBank and aligned separately (Babesia/Theileria over 582 bp; Ehrlichia over 618 bp; Rickettsia over 375 bp) with sequences obtained in this study, using ClustalW [44 (link)] in the software MEGA v7.00 [45 (link)]. The best-fit evolutionary models for each dataset were selected based on Corrected Akaikeʼs information criterion (cAIC) and Bayesian information criterion (BIC) using MEGA. Phylogenetic trees were constructed using the Neighbour-joining (NJ) and Maximum Likelihood (ML) methods in MEGA and Bayesian Inference (BI) method using Mr Bayes in Geneious Prime [46 (link)]. Each Bayesian analysis was run over 20,000,00 generations (ngen = 20,000,00) with two runs and every 400^th tree was saved (samplefreq = 400). For the NJ tree estimations, evolutionary distances were computed using the p-distance method whereas for the ML method, initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log-likelihood value. All positions containing gaps and missing data were eliminated. Bootstrapping method (10,000 replicates) was used to assess the reliability of internal branches and all trees were visualized and edited using MEGA. Plasmodium falciparum (GenBank: M19172), A. marginale (GenBank: AF414872) and R. bellii (GenBank: AY362703) were used as outgroups for Babesia/Theileria, Ehrlichia and Rickettsia, respectively.

Total DNA was extracted from the ticks by alkaline lysis as described elsewhere [57 (link)]. DNA extracts were stored at -20°C. The presence of the DNA of different tick-borne pathogenic species (Rickettsia spp., B. burgdorferi s.l., Ehrlichia/Anaplasma spp. and Babesia spp.) was determined by polymerase chain reaction (PCR) followed by reverse line blotting (RLB) as described before [52 (link),57 (link),58 (link)]. The probes that were used for RLB analysis of the PCR products can be found in Table 1.

PCRs were performed to characterize notable bacterial species detected in tick samples through the NGS screening. The bacteria Anaplasma, Ehrlichia, Bartonella, Coxiella, Francisella and Rickettsia were targeted in the PCR amplification and sequencing. The details of all primers used for the bacteria species identification are described in Table S1.
PCRs targeting the citrate synthase gene (gltA), which amplified a 694 bp fragment, and cell division protein gene (ftsZ), which amplified a 900 bp fragment of Bartonella, were conducted in semi-nested and single PCR, respectively. For Francisella species characterization, a fraction of the T-cell epitope gene (tul4) and 16S rDNA of Francisella were targeted in a single PCR to amplify 248 bp and 1 kb fragments, respectively. Single PCRs were conducted for Rickettsia species characterization by targeting six genes: gltA gene for 580 bp, outer membrane A gene (ompA) for 542 bp, outer membrane protein B gene (ompB) for 816 bp, 17 kDa common antigen gene (htrA) for 550 bp, 16S rDNA for 1.3 kb and surface cell antigen-4 gene (Sca4) for 928 bp fragment. All PCRs for Bartonella, Francisella and Rickettsia were conducted using Ex Taq Hot Start Version (Takara Bio) in a reaction mixture of 20 µl. The conditions used in the PCR assays were as follows: 35 or 40 cycles of denaturation at 94 °C for 30 s, annealing temperature according to each respective primer set for 30 s, and extension at 72 °C for 30 s, 60 s or 90 s depending on the targeted amplicon size.
Next, to characterize the species of Anaplasma, Ehrlichia and Coxiella, we used Tks Gflex DNA Polymerase (Takara Bio) with a 25 µl reaction mixture preparation. Nested PCR was conducted for Anaplasma and Ehrlichia by amplifying a 1.3 kb fragment of 16S rDNA of Anaplasmatacea with the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation step at 95 °C for 30 s, 48 or 54 °C of annealing for 30 s, and extension at 68 °C for 90 s, with a final extension at 68 °C for 5 min. A total of five genes were used for Coxiella species characterization, which included chaperone protein DnaK gene (dnaK) for 512 bp, chaperone protein GROEL gene (groEL) for 619 bp, β subunit of bacterial RNA polymerase gene (rpoB) for an estimate of 550 bp, 16S rDNA for an estimate of 1 kb and large ribosomal subunit (23S rDNA) for a 583–867 bp fragment. DNA of Coxiella was amplified with nested or semi-nested PCRs, with the following conditions: initial denaturation at 94 °C for 1 min, followed by 40 cycles of denaturation at 98 °C for 10 s, 54 or 56 °C of annealing for 15 s, and extension at 68 °C for 1 min, with a final extension at 68 °C for 5 min.
Finally, the amplicon size was verified with electrophoresis and visualized as described above. Sanger sequencing was performed on the successfully amplified samples using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems). The obtained sequencing products were analysed on an ABI Prism 3130X genetic analyzer (Applied Biosystems), as per the manufacturer’s instructions. The resulting sequences were assembled and trimmed using the ATGC software version 9.0.0 (GENETYX) and compared with the sequences available in the public databases using the Nucleotide Basic Local Alignment Search Tool (BLASTn) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

An enzyme-linked indirect immunosorbent assay (ELISA) was performed on sera samples for the detection of specific antibodies of L. infantum (INGEZIM® Leishmania; Ingenasa, Madrid, Spain), Ehrlichia canis (INGEZIM® Ehrlichia; Ingenasa) and Anaplasma phagocytophilum (Anaplasma-ELISA Dog; AFOSA GmbH, Blankenfelde-Mahlow, Germany). All assays were performed following the respective manufacturer’s instructions. All sera samples collected on all SDs were tested for all of the above pathogens.

The efficacy of the Seresto® (10% w/w imidacloprid/4.5% w/w flumethrin) collar was assessed based on the results of the various serological and molecular tests to detect exposure to CVBD pathogens (L. infantum, Ehrlichia spp., Anaplasma spp.) and ectoparasite counts (fleas and ticks) performed throughout the duration of the study.
A dog was considered positive for a given pathogen if exposure to the latter had been confirmed, either directly or indirectly, in one or more of the diagnostic tests. Dogs that tested negative for a particular or all CVBPs at study start were considered to be protected if the latter status was maintained until the final visit. The product was considered to be effective against fleas and ticks when the infestation intensity remained ≤ 1.
Only dogs that were followed up for at least 1 year (i.e. inclusion on SD 0 or SD 210 and at least 2 more time points of follow-up after inclusion) were included in the analyses. Dogs were excluded from the analyses if they tested positive in any of the CVBP tests performed at the day of inclusion. Dogs that tested positive in any of the tests at inclusion or/and during the study remained collared and were followed up like all other dogs.
Dogs were considered to be a treatment success if they had either no positive result in any of the follow-up tests or had only one positive ELISA result in any of the follow-up tests (the 1 positive result was considered to be a random positive test, possibly due to cross-reaction). Dogs were considered to be a treatment failure if they had either ≥ 2 positive ELISA results at follow-up time points, or a positive PCR result at the last evaluation time point.